Lipoproteins are the primary carriers of plasma cholesterol. They are micellar lipid-protein complexes (particles) having a surface film, comprised of one or more proteins associated with polar lipids, that surrounds a cholesterol-containing core. Lipoproteins were originally classified based on their buoyant densities as measured by ultracentrifugation. Accordingly, four major density classes have been recognized: chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins LDL and high-density lipoproteins (HDL).
Many studies have now established an inverse correlation between plasma HDL cholesterol levels and risk of coronary artery disease (CAD). That is, elevated levels of plasma cholesterol found in HDL particles correlate with a reduced risk of CAD.
Similarly, many studies have now shown that plasma levels of apolipoprotein AI (Apo AI), the major protein component of HDL, are also inversely related to the risk of CAD. In addition, Weisweiler et al., Clin. Chem., 27:348 (1981) have reported that knowledge of Apo AI levels may add to the predictive value of HDL cholesterol.
Because of its inverse correlation with CAD, there has been an extensive amount of research into the structure and function of Apo AI in lipid metabolism. Functionally, Apo AI is now believed to mediate the removal of cholesterol from tissues and to activate LCAT.
Structurally, purified Apo AI has been described as containing a high proportion (55%) of alpha-helix, which increases to 70% when it is associated with phospholipids as in the HDL particle. The lipid binding properties of Apo AI appear to be a function of a series of tandemly repeated segments of 22 amino acid residues punctuated mostly by proline residues that are alpha-helical and amphophilic.
The amino acid residue sequence of Apo AI, determined by Edman degradation of cyanogen bromide- and trypsin-fragments of intact Apo AI, has been described by Brewer et al., Biochem. Biophys. Res. Comm., 80:623-630 (1978). According to Brewer et al., cyanogen bromide (CNBr) cleavage of Apo AI produced four major fragments, designated CNBr1, CNBr2, CNBr3 and CNBr4, in order of their occurrence along the Apo AI sequence from amino-terminus to carboxy-terminus. Because it is of particular interest to the present invention, the amino acid residue sequence of the region of Apo AI from which CNBr2 is produced is illustrated in FIG. 1, along with the positions of the various fragments produced by trypsin cleavage of CNBr2.
It should be noted that CNBr2, like CNBr1, CNBr3 and CNBr4, is a polypeptide having homoserine lactone at its carboxy terminus as a result of the methione residue at that position being degraded during the CNBr cleavage process.
Immunochemical characterization of native Apo AI, i.e., Apo AI as it is found on HDL particles, has been problematical because it is antigenically heterogeneous and unstable. The antigenic heterogeneity of Apo AI appears to be the result of some epitopes being masked by lipids in the intact HDL or the antibody-binding ability of some epitopes being dependent on conformations of Apo AI as affected by lipids or other HDL associated proteins. The antigenic instability of Apo AI, as manifest by its changing immunoreactivity over time with defined antisera, appears to be due to such phenomena as self association and deamidation, both of which have been shown to occur in vitro.
Of particular interest to the present invention is deamidation, which, with Apo AI, results in the conversion of asparagine and glutamine residues into aspartic acid and glutamic acid, respectively. Deamidation of Apo AI can be accomplished in vitro by treatment with sodium hydroxide (NaOH), and is evidenced by its acquisition of a net negative charge, i.e., net increase in isoelectric point. See Curtiss et al., Proceeding of the Workshop on Lipoprotein Heterogeniety, Ed. by Lippel, National Institutes of Health Publication No. 87-2646, P. 363-377 (1987). According to Milthorp et al., Arterio., 6:285-296 (1986), the effects of storage and NaOH treatment on native Apo AI immunoreactivity are similar but not analogous, suggesting that while loss of Apo AI immunoreactivity during storage is due in large part to deamidation, more may be involved.
The antigenic heterogeneity and instability of Apo AI has made it difficult to produce assay systems for quantifying Apo AI in patient vascular fluid samples. This is because, inter alia, such systems require a reference material (standard) whose immunoreactivity for the system's primary anti-Apo AI antibody is consistent, at the very least, and preferably equivalent to that of the Apo AI in the patient's sample.
Recently, efforts at overcoming problems associated with the antigenic heterogenicity and instability of Apo AI have focused on using monoclonal antibodies (MAB) to identify epitopes on native Apo AI whose expression is consistent or "conserved" under specific isolation and storage conditions. Such epitopes, referred to herein as "conserved native epitopes", are further defined as Apo AI epitopes whose expression on HDL is not significantly affected, i.e., not significantly increased or decreased, as a result of processing or storage that results in deamidation.
An exemplary conserved native Apo AI epitope, designated epitope A, has been defined by Milthorpe et al., Arterio., 6:285-296 (1986) as being that portion of Apo AI CNBr1 that immunoreacts with MAB 4H1. According to Milthorp et al., the expression of epitope A remains constant overtime in patient serum samples stored at temperatures ranging from 4 degrees C. to -80 degrees C. This was in contrast to epitopes designated C, C' and C", all located in the CNBr2 region of Apo AI, and all of which were found to be "nonconserved" epitopes, i.e., epitopes whose expression was significantly increased or reduced upon storage at a similar range of temperatures.